Many of the most fundamental recombinant DNA operations involve gene isolation from recombinant DNA libraries, using radioactively labelled probes. The current procedures derive originally from the autoradiographic plaque screening methods of Benton, W. C. and Davis, R. W. (1977), Science 196, 180-182, as applied to recombinant DNA genome libraries (e.g., Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, G. K. and Efstratiadis, A. (1978), Cell 15, 687-701). As conventionally carried out sufficient plaques bearing individual recombinant phage are screened so that any given sequence will probably occur several times. For the human genome (3,000,000 kb genome size) an average of three occurrences requires about 500,000 different phage, while for sea urchin or Drosophila genomes the number is smaller (about 130,000 and 50,000 respectively) because of the smaller genome size. In present practice a library is propagated by growth in bacterial lawns on agar plates (often 155 mm in diameter). For each amplification or screening step the plaques are diluted and replated at about 1 phage per mm.sup.2. This is good practice since it prevents excessive loss of slower-growing phage by competition. A 500,000 phage library requires 25 plates of 20,000 mm.sup.2 area or about 0.5 m.sup.2 of bacterial lawn. In common practice the plaques are grown to nearly confluent lysis and the phage transferred to duplicate 155 mm diameter filters. The phage DNAs are then released by alkali and bound to the filters. The DNA matrix on the filter provides more or less faithful reproduction of the random array of plaques. After appropriate treatment the filters are hybridized with a radioactive probe, washed thoroughly, dried and autoradiographed under X-ray film. A radioactive spot occurring on both duplicates indicates the location of a recombinant phage plaque of interest. A plug containing this plaque and usually also the neighboring plaques is removed, diluted and replated. The filter transfer and hybridization process is repeated and finally the individual phage desired is selected and grown from one of the isolated positive plaques. Each time a library is screened a completely independent random set of plates is prepared.
Also involved are electrophoresis gels used for the sequencing of DNA, and blots transfered to filters and hybridized with a radioactive probe.
We have developed an apparatus which radically improves these procedures by direct radioactivity detection.
At present radioactive regions on filters and gels are detected by autoradiography with X-ray film (using intensifier screens). This method has the advantage that large areas can be examined at one time. The disadvantage is that one to several days of exposure are often required to detect typical spots that might contain 10 cpm distributed over 2 mm.sup.2. Such a spot could be reliably detected in a few minutes by directly counting the emitted beta particles. A direct counting method would only be practicable and advantageous if many spots were simultaneously counted over large areas. A rapid device for examining large areas to replace film autoradiography would be very useful for many purposes, particularly if it had a spatial resolution of less than 1 mm. Recent publications report the use of direct counting devices for examining small areas of chromatograms, but these methods are not applicable to large areas (Charpak, G., Melchart, G., Petersen, G. and Sauli, F. (1981). IEEE Trans. on Nucl. Sci. NS-28, 849-851; Aoyama, T. and Watanabe, T. (1978). Nucl. Instr. and Meth. 150, 203-208.) A spark chamber has been developed for high energy physics applications (Parkhomchuck, V. V., Pestov, Y. N. and Petrovykh, N. V. (1971). Nucl. Instr. and Meth. 93, 269-270; Atwood, W. B. (1980) Stanford Linear Accelerator Center - Pub. 2620 (Appendix B). This device consists of a thin gas filled region between two planar electrodes at a high DC voltage difference. The spark is limited (quenched) by a reduction in the electrode voltage and by the choice of gas. In many such devices the total counting rate is limited by the slow recovery (milliseconds) and sweeping out of ions. However, recently a major advance has been the introduction of high resistance semi-conducting glass for one of the electrodes. The charge on a local region of the glass is dissipated and then the voltage (locally) slowly rises in the same period as the ions are swept out. Carefully chosen organic gases absorb the UV light produced and prevent the spark from spreading to nearby still charged regions of the glass. Thus while local regions are "dead" for a few milliseconds after a spark the remainder is operational and the total counting rate may be high. Even in one region rates of many thousands of events per minute can be counted without loss. The sparks are detected by means of a relatively large electrical pulse (as much as 1 volt) they produce on metal strips placed outside the chamber on the upper surface of the glass. These strips are connected to amplifying circuits capable of precise time difference and/or pulse amplitude measurements.
The spark chamber already developed at Stanford Linear Accelerator Center includes a fast digital clock (TDC) for each upper external conducting strip as well as all of the associated electronics for digitization of the signals and computer linkage.
The chamber of this invention differs from the one described in having a thin metal or metal coated window for the lower (high voltage) electrode. The results of Atwood show that the low gas pressure and 1 mm spacing will be effective for the present use (Santonico, R. and Cardarelli, R. (1981). Nucl. Instr. and Meth. 187, 377-380; and Atwood, W. B. (1980) Stanford Linear Accelerator Center - Pub. 2620 (Appendix B) and personal communication). The lower window requirements are that it be smooth so as not to induce sparks at irregularities, and that the spacing between it and the glass be relatively uniform. Measurements indicate that an aluminum window of 0.2 mm thickness would only absorb about 15% of the .sup.32 P beta particles. A window in this range of thickness made of a high stiffness aluminum alloy or a copper coated epoxy fiberglass sheet meets all of the requirements.
Among the differences between the prior art and the present invention is precise timing of events. In the present invention, it is unimportant when the events take place and as a result it is possible to reduce the voltage, which reduces the critical requirements for smoothness of the window electrode and for uniformity of spacing. For the same reason it is now possible to work near atmospheric pressure (where time delays are greater), which is a large convenience in construction and sealing of the counter. Even at lower voltage and pressure the sparks themselves rise very abruptly and apparently the spatial resolution depending on time differences at the ends of the strips is not degraded.
It is believed that the present invention is a major advance in the art and it is to be expected that it will be widely adopted.